the noncommensal bacterium methylococcus capsulatus (bath ... · the noncommensal bacterium...

The Noncommensal Bacterium Methylococcus capsulatus (Bath)Ameliorates Dextran Sulfate (Sodium Salt)-Induced Ulcerative Colitisby Influencing Mechanisms Essential for Maintenance of the ColonicBarrier Function

Charlotte R. Kleiveland,a Lene T. Olsen Hult,a Signe Spetalen,c Magne Kaldhusdal,d Trine Eker Christofferesen,a,e Oskar Bengtsson,a

Odd Helge Romarheim,f Morten Jacobsen,b Tor Leaa

Department of Chemistry, Biotechnology and Food Science, The Norwegian University of Life Sciences, Ås, Norwaya; Ostfold Hospital Trust, Fredrikstad, Norwayb; OsloUniversity Hospital, The Norwegian Radium Hospital, Oslo, Norwayc; Norwegian Veterinary Institute, Oslo, Norwayd; Faculty of Engineering, Østfold University College,Halden, Norwaye; Department of Animal and Aquacultural Sciences, Aquaculture Protein Centre, CoE, Norwegian University of Life Sciences, Ås, Norwayf

Dietary inclusion of a bacterial meal has recently been shown to efficiently abolish soybean meal-induced enteritis in Atlanticsalmon. The objective of this study was to investigate whether inclusion of this bacterial meal in the diet could abrogate diseasedevelopment in a murine model of epithelial injury and colitis and thus possibly have therapeutic potential in human inflamma-tory bowel disease. C57BL/6N mice were fed ad libitum a control diet or an experimental diet containing 254 g/kg of body weightBioProtein, a bacterial meal consisting of Methylococcus capsulatus (Bath), together with the heterogenic bacteria Ralstonia sp.,Brevibacillus agri, and Aneurinibacillus sp. At day 8, colitis was induced by 3.5% dextran sulfate sodium (DSS) ad libitum in thedrinking water for 6 days. Symptoms of DSS treatment were less profound after prophylactic treatment with the diet containingthe BioProtein. Colitis-associated parameters such as reduced body weight, colon shortening, and epithelial damage also showedsignificant improvement. Levels of acute-phase reactants, proteins whose plasma concentrations increase in response to inflam-mation, and neutrophil infiltration were reduced. On the other, increased epithelial cell proliferation and enhanced mucin 2(Muc2) transcription indicated improved integrity of the colonic epithelial layer. BioProtein mainly consists of Methylococcuscapsulatus (Bath) (88%). The results that we obtained when using a bacterial meal consisting of M. capsulatus (Bath) were simi-lar to those obtained when using BioProtein in the DSS model. Our results show that a bacterial meal of the noncommensal bac-terium M. capsulatus (Bath) has the potential to attenuate DSS-induced colitis in mice by enhancing colonic barrier function, asjudged by increased epithelial proliferation and increased Muc2 transcription.

Inflammatory bowel disease (IBD) manifests itself by chronicinflammation characterized by acute flares followed by periodsof remission. The etiology of IBD remains incompletely under-stood, but it is generally accepted that a complex interplay be-tween genetic, environmental, and immunological factors con-tributes to disease initiation and progression (1, 2). Crohn’sdisease (CD) and ulcerative colitis (UC) are clinically the mostserious manifestations of IBD; however, inflammation is fre-quently present in microscopic colitis, collagen colitis, and evenirritable bowel syndrome (IBS), which indicates the central role ofinflammation in intestinal homeostasis.

The critical function of the intestinal epithelial cells and themucus layer is to form a barrier that separates luminal contentsfrom the lamina propria. Loss of epithelial barrier function fol-lowed by invasion of bacteria is thought to be an initial event thatprovokes injury and inflammation in many intestinal disorders,including IBD (3, 4).

The cross talk between intestinal epithelial cells (IECs), im-mune cells, and intestinal bacteria represents a fundamental fea-ture in maintaining normal intestinal homeostasis and for mount-ing protective immune responses against pathogens (5, 6).Accumulating evidence indicates that recognition of intestinalbacteria through pattern recognition receptors (PRRs) is impor-tant for controlling intestinal homeostasis and that the context ofPRR activation is critical for the outcome (7). Basal PRR activationis essential for maintaining the barrier function and the composi-

tion of the microbiota in a healthy intestine, while aberrant PRRsignaling may be a contributor to the development of IBD (8, 9).The association between the Nod-like receptor nucleotide-bind-ing oligomerization domain-containing protein 2 (NOD2) andCD is well established. It has also been shown that mice lackingspecific Toll-like receptors (TLRs) or that are deficient in theshared TLR signaling protein myeloid differentiation primary re-sponse gene 88 (Myd88) have increased susceptibility to dextransulfate sodium (DSS)-induced colitis, characterized by defectivetissue repair and increased mortality (10). A possible link betweenpolymorphisms in C-type lectin receptors and IBD has also re-cently been reported (11). PRRs recognize conserved patterns indiverse microbial molecules, so-called microbe-associated micro-bial patterns (MAMPs). Such MAMPS bind to PRRs with differ-ent avidities, eliciting different intracellular activation events, andcombinations of MAMPs may also act in both synergistic andantagonistic ways to modulate cellular responses (12). Tradition-

Received 10 August 2012 Accepted 1 October 2012

Published ahead of print 12 October 2012

Address correspondence to Charlotte R. Kleiveland, [email protected].

C.R.K. and L.T.O.H. contributed equally to this article.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.02464-12

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ally, studies of microbe-host interactions are carried out with dif-ferent pathogens and natural commensals. Thus, both commen-sals and probiotic bacteria have been demonstrated to mediateanti-inflammatory effects in different mouse models of IBD (13–15). However, the vast number of noncommensals may representan unrecognized source of MAMPs for regulation of immune andinflammatory responses. To our knowledge, no one has tried toinvestigate and exploit the potential of noncommensals in modu-lating the inflammatory process in IBD.

Romarheim et al. (16) recently reported that dietary inclusionof a bacterial meal (BioProtein) could prevent the development ofsoybean meal-induced enteritis in Atlantic salmon. BioProtein is abacterial meal obtained by aerobic fermentation of natural gas byMethylococcus capsulatus (Bath), an obligate methanotroph (17),together with the heterogenic bacteria Ralstonia sp., Brevibacillusagri, and Aneurinibacillus sp., representing minor fractions of thepreparation. Thus, this bacterial meal includes a variety of li-gands for different PRRs. We wanted to extend the observa-tions made in salmonids to mammals and exploited the well-established model of DSS-induced colitis in mice. Clinicalevaluation of DSS-exposed mice fed a standard diet including25% bacterial meal revealed that the mice had a strikingly en-hanced well-being compared to DSS-exposed mice fed thestandard diet. These observations were confirmed by morpho-logical data demonstrating a significant reduction of inflam-mation and colonic tissue damage in mice receiving a bacterialmeal-containing diet. We also observed increased epithelialcell proliferation and increased colonic crypt depths in micefed the bacterial meal. All findings were confirmed in a exper-iment where C57BL/6N mice were fed a standard diet including25% Methylococcus capsulatus (Bath) only. These observationssuggest that M. capsulatus, a bacterium not recognized to be agut commensal, may provide new and unique opportunities forstudying mechanisms involved in the development and treat-ment of IBD and how microbe-host interactions can be ex-ploited to manipulate the inflammatory tonus of the intestinalmucosa.

MATERIALS AND METHODSAnimals. Female C57BL/6NTac mice (age, 5 to 6 weeks; weight, 15 to 18g) (18) from Taconic (Ry, Denmark) and with conventional microbiolog-ical status were used. The animals were divided into four groups with sixanimals per group. The animals were fed ad libitum a control diet based onAIN-93G (19) or an experimental diet where the casein content (200 g/kgof body weight) and corn starch (54 g/kg) were exchanged with 254 g/kgBioProtein (Norferm AS, Stavanger, Norway) or freeze-dried M. capsula-tus (Bath). Two groups of animals were fed the control diet and the othertwo were fed the experimental diet for 2 weeks. The animals were accli-matized to the diet for 7 days before induction of colitis. At day 8, colitiswas induced by 3 to 3.5% DSS (TdB Consultancy AB, Uppsala, Sweden)ad libitum in the drinking water for 6 days. The mice were sacrificed at day6 following DSS treatment.

Tissue collection. The colon from the cecum to the rectum was col-lected, and the length of the colon was measured. The content of the colonwas washed out with phosphate-buffered saline before it was openedalong the length and prepared in a Swiss roll format. The Swiss roll wasimmediately frozen in liquid nitrogen and then stored at �80°C. Thelength of the colon was measured.

RNA extraction and cDNA synthesis. Twenty to 30 mg of colonictissue and 600 �l RLT lysis buffer (Qiagen, Germantown, MD) with mer-captoethanol were placed in an M tube (Miltenyi GmBH, BergischGladbach, Germany). The tissue was dissociated by a gentleMACS disso-

ciator (program RNA_02; Miltenyi GmBH, Bergisch Gladbach, Ger-many). RNA was extracted according to the manual for the QiagenRNeasy kit (Qiagen, Germantown, MD). DNase treatment was includedin the protocol. RNA was eluted in 30 �l RNase-free water and stored at�80°C. The RNA concentration was obtained from A260 measurementsusing a NanoDrop 2000 spectrophotometer (Thermo Fischer ScientificInc., Waltham, MA). The RNA integrity number (RIN) was tested by anAgilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA) andranged from 6.5 to 8. cDNA synthesis was performed by a SuperScriptVILO cDNA synthesis kit (Life Technologies Ltd., Paisley, United King-dom) according to the manufacturer’s recommendations (2.3 �g RNA/reaction mixture). The cDNA synthesis program was 25°C for 10 min,42°C for 120 min, and 85°C for 5 min with a final hold at 4°C (MJ Researchthermocycler, Watertown, MA).

Real time-PCR. PCR was performed using a TaqMan gene expressionmaster mix (Applied Biosystems, Carlsbad, CA), according to the manu-facturer’s recommendations, with 20 ng cDNA per reaction mixture. AllPCR mixtures were amplified by a Rotor Gene 6000 real-time PCR ma-chine (Qiagen, Germantown, MD). The following PCR program wasused: 50°C for 2 min, 95°C for 5 min, 95°C for 15 s, and 60°C for 1 min (40cycles of the two last steps). All primer/probe sets used were TaqMan geneexpression assays (Applied Biosystems, Carlsbad, CA). Rpl32 was identi-fied as the most stable reference gene using a mouse geNorm kit (Prim-erdesign, Southampton, United Kingdom) and geNorm software.

The following TaqMan probes were used in this study: Mm00441203_m1for serum amyloid A3 (Saa3), Mm00656925_m1 for S100a9, Mm00447886_m1for myeloperoxidase (MPO), Mm00458293_g1 for mucin 2 (Muc2),Mm00440502_m1 for nitric oxide synthase 2 (NOS2), Mm02528467_g1for Rpl32, Mm01336189_m1 for interleukin-1� (IL-1�), Mm00446190_m1 forinterleukin-6, Mm00439614_m1 for interleukin-10, and Mm00439618_m1 forinterleukin-17A.

Histology. Frozen tissue samples were mounted in Tissue-Tekmounting medium (Sakura, Torrance, CA) in a cryostat at �20°C. Sec-tions (8 �m) were obtained at �20°C, and collected on polylysine-coatedglass slides (Thermo Fischer Scientific Inc., Waltham, MA). Sections werebriefly dried at room temperature, before they were postfixed in formalinfor 1 min. The slides were then stained with hematoxylin-erytrosin (HE).

Histological scoring of the colitis was done blindly by an independentpathologist according to the scoring system presented in Table 1 (20).

For immunohistochemistry, the sections of paraffin-embedded tissuewere incubated for 1 h at room temperature with blocking solution, 5%rabbit serum in Tris-buffered saline–Tween 20 (TBS-T) buffer, before ratKi67 (clone MM1) antibody solution was added (Monosan). Incubationwith primary antibody was performed overnight at 4°C. Primary antibodysolution was removed, and sections were washed briefly in TBS-T bufferbefore rabbit anti-rat fluorescein isothiocyanate antibody solution wasadded (Jackson ImmunoResearch). Sections were incubated with second-ary antibody for 1 h at room temperature in the dark. Sections were againwashed in TBS-T buffer, before the nuclear stain Hoechst 3342 was addedfor 10 min at room temperature (Sigma-Aldrich, St. Louis, MO). Slides

TABLE 1 Scoring system for inflammation-associated histologicalchanges in the colona

Score Histological change

0 No evidence of inflammation1 Low level of inflammation with scattered infiltrating mononuclear

cells (1–2 foci)2 Moderate inflammation with multiple foci3 High level of inflammation with increased vascular density and

marked wall thickening4 Maximal severity of inflammation with transmural leukocyte

infiltration and loss of goblet cellsa Slides were scored by a pathologist and blindly analyzed concerning experimentalgrouping.

Bacterial Meal Ameliorates DSS-Induced Colitis

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were mounted either with immersion oil or ProLong Gold antifade re-agent (Life Technologies Ltd., Paisley, United Kingdom) before visualiza-tion with a Leica SP5 confocal microscope (Leica Microsystems, Wetzlar,Germany).

Cultivation of bacteria and preparation of M. capsulatus bacterialmeal. Methylococcus capsulatus (Bath) NCIMB 11132 was used to producesingle-strain bacterial meal. Nitrate mineral salts (NMS) medium (21)was used for all types of cultivations. M. capsulatus (Bath) culture aliquotswere frozen in liquid nitrogen and stored at �80°C. Cultivations on agarplates and in shake flasks were performed at 45°C in an atmosphere of75% air, 23.25% CH4, and 1.25% CO2. Shake flask cultures were incu-bated in an orbital shaker incubator at 200 rpm. Continuous cultivationwas carried out in a 3-liter bioreactor (ADI Autoclavable bioreactor sys-tems; Applikon, Schiedam, The Netherlands) with a working volume of 2liters. Cells precultivated in shake flasks were used to inoculate the biore-actor to an optical density at 440 nm (OD440) of 0.1. The temperature wasmaintained at 45°C, stirring was set to 650 rpm, and a culture pH of 6.8was maintained by automatic addition of 2.5 M NaOH. A gas mixture of55% air, 24% methane, 20% O2, and 1% CO2 was sparged into the biore-actor at a constant flow of 0.064 volume of gas per volume of liquid andminute (vvm). The continuous culture was started after an initial batchphase, and the dilution rate was set to 0.01 h�1. The OD440 at steady statewas generally sustained at approximately 10. Culture effluent was col-lected, and cells were harvested by centrifugation. Bacterial cell walls weredisrupted by the use of a French press before freeze-drying of the material.

Statistics. The results were expressed as the means � standard devia-tions or with a range with an upper and lower value for exponential values.

Experiments were conducted at least in duplicate, and similar results wereobtained. The differences between means were tested for statistical signif-icance using one-way analysis of variance (ANOVA). A P value of �0.05was considered statistically significant in all experiments.

RESULTSOral administration of bacterial meal to C57BL/6 mice reduceddisease activity and histological inflammation in acute DSS-in-duced colitis. Administration of DSS to C57BL/6 mice induces anacute inflammation of the colon, pronounced weight loss, andbloody diarrhea. In this study, C57BL/6 mice fed the standard dietand given 3.5% DSS in the drinking water developed progressiveweight loss (Fig. 1A) with diarrhea and blood in the feces. How-ever, the symptoms of DSS treatment were less profound or min-imized after pretreatment with the standard diet containing thebacterial meal. The increase in body weight of DSS-treated micefed a bacterial meal-containing diet was comparable to that of thecontrol group (5.4% � 2.6% and 5.6% � 2.5% mean increases inbody weight, respectively), while DSS-treated mice eating thestandard diet experienced a significant weight loss (�7.9% �11.1%; P � 0.001) In addition, the DSS-treated mice in the stan-dard diet group exhibited a significant shortening of the coloncompared to the control group (�1.6 � 0.3 cm; P � 0.001), whilethe length of the large intestine of the bacterial meal-pretreatedmice did not deviate significantly from that of the large intestine of

FIG 1 (A) Weight of the mice followed during treatment with DSS, presented as change in weight from day 1 (in percent). (B) Colon length measured directlyafter dissection from the mice before emptying of the contents. (C) Histological scoring. Upon histological examination, the severity of the inflammation wasscored on a scale of from 0 to 4, with 4 being the most severe. Data are presented as mean � standard deviations. Differences between means were tested byone-way ANOVA. A P value of �0.05 was considered statistically significant (*). SD, standard diet; BM, bacterial meal.

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the control group (�0.4 � 0.9 cm) (Fig. 1B). On histologicalexamination of colons from mice in the control group, a typicalnormal structure was found with a mean histological score of0.7 � 0.5. In DSS-treated mice receiving the standard diet, theaverage histological score was 3.2 � 0.4, which was significantlyhigher than that for DSS-treated mice receiving a diet containingbacterial meal, which had an average score of 2.3 � 0.5 (P � 0.05).Mice fed a bacterial meal diet had a mean histological score of1.0 � 0, which was comparable to that for the group fed the stan-dard diet (Fig. 1C and Fig. 2).

Inclusion of bacterial meal in the diet decreased intestinalinflammatory markers in mice with DSS-induced colitis. To as-sess inflammatory activity, expression of the genes for two well-known diagnostic markers of IBD, S100a9 (calprotectin) and se-rum amyloid A3 (Saa3), in the colon was determined (22, 23). ThemRNA levels of both S100a9 and Saa3 were found to be upregu-lated after treatment with DSS in mice fed the standard diet(1,350.2-fold [range, 478- to 3,822-fold; P � 0.0001] and 64-fold[range, 28- to 147-fold; P � 0.001] for S100a9 and Saa3, respec-tively). However, DSS-induced expression of the genes for S100a9and Saa3 was lower in mice fed a diet containing bacterial meal(84-fold [range, 32- to 223-fold; P � 0.001] and 14.9-fold [range,

7- to 32-fold]) than animals in the DSS control group, althoughnot significantly so for Saa3 (Fig. 3A).

Neutrophil infiltration and activity were lower in mice fed abacterial meal. DSS-induced colitis is characterized by infiltrationof immune cells in the mucosa and submucosa, epithelial ulcer-ation, and crypt damage (24, 25). The level of MPO has beenshown to be proportional to the number of neutrophils in theinflamed tissue (26). Here, we found that the mRNA level forMPO in the colon was significantly increased in both DSS-treatedgroups compared to that in the control group (4.6-fold [range,3.2- to 6.5-fold; P � 0.001] and 2.1-fold [range, 1.5- to 3.0-fold;P � 0.001] for mice fed the standard diet and bacterial meal,respectively). However, MPO mRNA expression decreased bymore than 50% in DSS-treated mice receiving the bacterial mealcompared to that in the group receiving the standard diet, al-though the difference was not statistically significant (Fig. 3B).MPO activity has been strongly associated with colonic tissue in-jury resulting from DSS administration in inducible NOS2�/�

mice, suggesting that NOS2 is important for the signaling processin neutrophils (27). After DSS treatment, there was a significantincrease in NOS2 mRNA transcription in the colons of both micefed the standard diet and mice fed the bacterial meal (78.8-fold

FIG 2 (1 and 2) HE-stained paraffin-embedded section of colon from a mouse fed a standard diet; (3 and 4) HE-stained paraffin-embedded section of colonfrom a mouse fed a standard diet and exposed to DSS in the drinking water; (5 and 6) HE-stained paraffin-embedded section of colon from a mouse fed aBioProtein bacterial meal; (7 and 8) HE-stained paraffin-embedded section of colon from a mouse fed a BioProtein bacterial meal and exposed to DSS in thedrinking water.

FIG 3 (A) mRNA levels for S100a9 (calprotectin) and Saa in DSS-treated mice given a standard diet or a bacterial meal relative to those in DSS-untreated micegiven a standard diet. mRNA levels were determined by real-time PCR. (B) mRNA levels for MPO and NOS2 in DSS-treated mice given a standard diet or abacterial meal relative to those in DSS-untreated mice given a standard diet. mRNA levels were determined by real-time PCR. Data are presented as the mean foldchange � standard deviation. Differences between means were tested by one-way ANOVA. A P value of �0.05 was considered statistically significant (*).




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[range, 51- to 119.4-fold; P � 0.001] and 22.6-fold [range, 16- to32-fold; P � 0.001], respectively). The NOS2 mRNA transcriptlevel was, however, approximately 70% lower in the colons ofDSS-induced mice receiving bacterial meal than the colons ofmice fed the standard diet (3.5-fold; range, 2.3- to 5.3-fold; P �0.01) (Fig. 3B).

In the colons of DSS-treated mice fed a bacterial meal, therewas a lower induction of cytokine production. The immunolog-ical effects of the bacterial meal-containing diet could be respon-sible for the clinical improvement seen in the mice. Further insightwas provided by analyzing the levels of cytokines in colonic tissue.The mRNA transcript levels of IL-1�, IL-6, IL-10, and IL-17 in thecolon were investigated. There was a lower induction of mRNA ofall cytokines in DSS-treated mice receiving bacterial meal than inanimals fed the standard diet. The proinflammatory cytokineIL-1� was upregulated in both DSS-treated groups (45.3-fold[range, 26- to 78.8-fold; P � 0.001] and 7.2-fold [range, 4.9- to10.6-fold; P � 0.001]) for mice fed the standard diet and bacterialmeal, respectively), but there was a 2.6-fold lower induction ofIL-1� in animals receiving bacterial meal. IL-6 was upregulated inboth DSS groups (256-fold [range, 128- to 512-fold; P � 0.001]and 104-fold [range, 55.7- to 194-fold; P � 0.001] for mice fed thestandard diet and bacterial meal, respectively), but we found a2.5-fold lower increase in the induction of IL-6 in the group givena bacterial meal than the group given the standard diet. IL-10 wasupregulated in both DSS-treated groups (14.9-fold [range, 11.3-to 19.7-fold; P � 0.001] and 2.6-fold [range, 2- to 3.4-fold; P �0.05] for mice fed the standard diet and bacterial meal, respec-tively), but we found a 5.7-fold (P � 0.001) lower increase in theinduction of IL-10 in the group given a bacterial meal than thegroup given the standard diet. IL-17 was upregulated in both DSSgroups (168.9-fold [range, 48.5- to 588.1-fold; P � 0.01] and 84.4-fold [range, 26- to 274.3-fold; P � 0.01] for mice fed the standarddiet and bacterial meal, respectively), but we found a 2-fold lowerincrease in the induction of IL-17 in the group given a bacterialmeal than the group given the standard diet (Fig. 4A). This reduc-tion in cytokines indicates a reduced induction of inflammation inthe colonic tissue.

The bacterial meal-containing diet led to a slight increase inMuc2 mRNA expression. An intact inner mucous layer is impor-tant in the protection of the colon epithelial cells, as bacteria in

contact with the epithelium are likely to trigger an inflammatoryresponse. This mucous layer is formed by the gel-forming mucinMuc2, which is the major secretory mucin synthesized and se-creted by goblet cells (28). Gene expression levels of Muc2 werefound to be decreased in DSS-treated mice given the control diet(�2-fold [range, �2.6- to �1.5-fold]). On the other hand, DSS-treated mice which received bacterial meal showed a slight in-crease in Muc2 gene expression (1.3-fold [range, 1- to 1.7-fold])compared to mice that received the control diet. There was a sig-nificant difference in Muc2 mRNA expression between DSS-treated mice receiving the standard diet and those receiving bac-terial meal (2.6-fold [range, 2- to 3.5-fold; P � 0.05]) (Fig. 4B).Goblet and absorptive cells also express membrane-bound mu-cins Muc3 and Muc4, which are components of the glycocalyx,but only slight changes in Muc3 and Muc4 gene expression werefound (results not shown).

Mice fed the bacterial meal diet showed increased cryptdepths and a higher number of proliferating epithelial cells. Thedepth of the colon crypts of mice fed a diet containing the bacterialmeal for 14 days was increased compared to that for mice fed thestandard diet. Crypt depth reflects the number of epithelial cellspresent in the crypt. Colon tissue sections were stained withHoechst 3342 to visualize the nucleus of all cells, and then thenumber of cells in each crypt was counted. The numbers of epi-thelial cells in 20 crypts per animal in each of the two groups werecounted. The average numbers of cells per crypt were 25.5 � 0.6and 31.4 � 0.18 for mice fed the standard diet and bacterial meal,respectively (P � 0.001) (Fig. 5A).

To investigate whether the increase in the number of cells wascaused by increased proliferation of the crypt cells, paraffin-embedded tissue was stained with anti-Ki67 antibodies. AntigenKi67 is a nuclear protein associated with cellular proliferation(29). There was an obvious increase in the number of Ki67-posi-tive cells in the crypts of animals fed a bacterial meal diet com-pared to that in the crypts of animals fed the standard diet. TheKi67-positive cells were found to be higher up in the crypts in micefed the bacterial meal than in mice fed the standard diet (Fig. 5B).

Oral administration of a bacterial meal of M. capsulatus(Bath) reduced disease activity and histological inflammation inacute colitis. M. capsulatus (Bath) is the main constituent of Bio-Protein, and therefore, an animal study was conducted with a diet

FIG 4 (A) mRNA levels for the cytokines IL-1�, IL-6, IL-10, and IL-17 in DSS-treated mice given a standard diet or a bacterial meal relative to those inDSS-untreated mice given a standard diet. mRNA levels were determined by real-time PCR. (B) mRNA level for Muc2 in DSS-treated mice given a standard dietor a bacterial meal relative to that in DSS-untreated mice given a standard diet. mRNA levels were determined by real-time PCR. Data are presented as the meanfold change � standard deviation. Differences between means were tested by one-way ANOVA. A P value of �0.05 was considered statistically significant (*).

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containing a bacterial meal of M. capsulatus (Bath). C57BL/6 micefed the standard diet and exposed to 3.0% DSS in the drinkingwater developed progressive weight loss (Fig. 6A) with diarrheaand blood in the feces. Animals pretreated with a diet containingM. capsulatus meal showed significantly fewer symptoms afterDSS treatment. The body weight of DSS-treated mice fed the M.capsulatus-containing diet was comparable to that of mice in thecontrol group (19.3 � 0.7 g and 19.3 � 0.7 g, respectively), whileDSS-treated mice eating the standard diet experienced a signifi-cant weight loss (17.1 � 0.9 g; P � 0.001). On histological exam-ination of colons from mice in the control group, a typical normalstructure with a mean histological score of 0 was found. In DSS-treated mice receiving the standard diet, the average histologicalscore was 3.2 � 1.1, which was significantly higher than that forDSS-treated mice fed a diet containing M. capsulatus meal, whichhad an average histological score of 1.8 � 1 (P � 0.01). Mice fed anM. capsulatus-containing diet had a mean histological score of 0,

which was the same as that for the control group (Fig. 6B and 7).These findings confirm that M. capsulatus (Bath) represents theactive component of the bacterial meal BioProtein.

DISCUSSION

The objective of this study was to investigate whether inclusion ofthe bacterial meal BioProtein in the diet could abrogate disease ina murine model of epithelial injury and colitis and thus have ther-apeutic potential in human IBD. This bacterial meal has recentlybeen reported to attenuate soybean meal-induced enteritis in At-lantic salmon (16). Although the mechanism is unknown, it islikely that the enteritis in salmonids is a T-cell- and cytokine-mediated inflammation (30, 31) and thus shows similarities withIBD in mammals.

To evaluate the effect of this bacterial meal, we used the well-established model of DSS-induced colitis in mice (32). DSS-in-duced colitis exhibits several characteristics resembling human

FIG 5 (A) Number of cells lining the lumen of the colon crypts in mice fed a standard diet (1 and 2) or a bacterial meal (3 and 4). The number of cells is presentedas the mean per crypt � standard deviation. The cell number was counted in 20 crypts per animal. (B) Paraffin-embedded sections of representative colon cryptsfrom animals fed a standard diet (1 and 2) or a bacterial meal (3 and 4) stained with anti-Ki67 antibodies and Hoechst 3342. Crypt structures are indicated bywhite lines.




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ulcerative colitis, including weight loss, severe diarrhea, rectalbleeding, ulceration and loss of epithelium, and leukocyte infiltra-tion (27, 32, 33). Notably, the bacterial meal-containing diet sig-nificantly improved the clinical, colitis-associated findings of re-duced body weight, shortening of the colon, and epithelialdamage.

The presence of active gut inflammation in IBD is associatedwith an acute-phase reaction and migration of leukocytes to thegut, which is translated into enhanced expression of acute-phaseproteins like Saa3 (23). The level of Saa3 mRNA was significantlyincreased in both DSS-treated groups but was greatly reduced inanimals fed the bacterial meal. This indicates a lower disease ac-tivity in animals fed a bacterial meal-containing diet than animalsfed a control diet.

It is well accepted that in many inflammatory disorders of theintestine, the combination of epithelial injury, disease activity, and

symptoms parallel neutrophil infiltration of the mucosa (34, 35).Neutrophil accumulation also plays an important role in DSS-induced colitis, as the disease can be attenuated by administrationof neutrophil antiserum (36), and the level of MPO is reported tobe proportional to the number of neutrophils in inflamed tissue(26). We found that the mRNA expression level of the neutrophilmarker MPO was reduced by more than 50% in bacterial meal-fedanimals, further supporting reduced levels of tissue inflammation.The strong association between MPO expression and tissue injuryin DSS-treated NOS2�/� mice (27) indicates that NOS2 is impor-tant for neutrophil function. Adding bacterial meal to the dietsignificantly reduced the level of NOS2 mRNA in DSS-treatedanimals. Calprotectin (S100a9) represents 60% of the cytosolicproteins in granulocytes. The levels of S100a9 in the lamina pro-pria tend to mirror the level of neutrophil migration to the gas-trointestinal tract. S100a9 mRNA levels are also significantly re-

FIG 6 (A) Weight of the mice fed a diet containing a Methylococcus bacterial meal (MM) followed during the period of treatment with DSS; (B) colon lengthmeasured directly after dissection from the mice before emptying of the contents.

FIG 7 (1 and 2) HE-stained paraffin-embedded section of colon from a mouse fed a standard diet; (3 and 4) HE-stained paraffin-embedded section of colonfrom a mouse fed a standard diet and exposed to DSS in the drinking water; (5 and 6) HE-stained paraffin-embedded section of colon from a mouse fed aMethylococcus bacterial meal; (7 and 8) HE-stained paraffin-embedded section of colon from a mouse fed a Methylococcus bacterial meal and exposed to DSS inthe drinking water.

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duced in bacterial meal-fed animals compared to those in mice feda control diet. Hence, the significant reduction in markers of neu-trophil granulocytes clearly shows that inclusion of a bacterialmeal in the diet results in lower disease activity and less inductionof gastrointestinal inflammation in response to DSS treatment.

The cytokines produced in the gut mucosa tend to greatly in-fluence the immunological outcome. Production of anti-inflam-matory cytokines induces mucosal unresponsiveness and toler-ance, while production of proinflammatory cytokines indicates aprotective immune response and inflammation. Prophylactictreatment with the bacterial meal before DSS exposure decreasedthe production of proinflammatory cytokines IL-6, IL-1�, andIL-17, as well as the anti-inflammatory cytokine IL-10, in the co-lon. This indicates that prophylactic feeding of the bacterial mealto the animals strongly reduced the induction of inflammation inthe colon, as all inflammatory markers were less induced than theywere in the mice fed the control diet. Furthermore, reduced IL-10mRNA levels also indicate lowered T-cell activation, in general,suggesting enhanced barrier function and less activation of adap-tive immunity in bacterial meal-fed mice.

To protect the large intestine from the enormous amounts ofcommensal bacteria, a mutualistic relationship has evolved whereboth host and bacteria benefit. An inner, firmly adherent mucouslayer and an outer, loose nonadherent mucous layer have beenidentified (37, 38). Although the molecular compositions of thesetwo layers are similar, consisting mainly of Muc2, the bacterialflora resides in the loose mucus, whereas the inner firm layer ofmucus is impervious to bacteria and thereby functions as a pro-tective barrier for the epithelial cell surface (38). DSS treatmentresulted in decreased Muc2 mRNA transcription, whereas pro-phylactic intake of a bacterial meal-containing diet before DSStreatment significantly enhanced Muc2 mRNA transcription.This difference in Muc2 production could be of importance inmaintaining epithelial layer integrity in the colons of mice fed abacterial meal-containing diet.

An important part of the intestinal regenerative response isstimulation of epithelial cell proliferation, as this will supply cellsfor the epithelium and promote and maintain the crypt and villusdevelopment of the regenerating mucosa. Epithelial cell prolifer-ation is increased in crypts adjacent to injured areas with bothcrypt elongation and crypt fission (39). Mice fed the bacterialmeal-containing diet showed a significant increase in the cryptdepth of the colon. The number of crypt cells and the number ofKi67-positive proliferating cells were increased in animals eatingthe bacterial meal-containing diet. This also resulted in a modestincrease in the colon length of these animals. Increased epithelialcell proliferation in response to the bacterial meal was also iden-tified in Atlantic salmon by Romarheim et al. (16). Enhancedepithelial cell proliferation and, hence, increased epithelial cellregeneration could be essential for the reduced disease activityseen after inclusion of bacterial meal in the diet. The increased rateof regeneration could make the epithelial cell layer in the intestinemore resistant to damage and thereby less susceptible to inductionof inflammation.

An animal study with inclusion of a bacterial meal from M.capsulatus (Bath) verifies only that the active component(s) ofBioProtein leading to amelioration of colitis is derived from thisbacterium.

The Gram-negative bacterium M. capsulatus (Bath) has so farnot been reported to be a part of the human microbiota. The

bacteria are lysed by high pressure and then spray dried (40), andtherefore, the bacterial meal does not contain any live bacteriacapable of colonizing the intestine. However, the preparation con-tains a multitude of bacterial components with potential to inter-act with cells of the epithelium and lamina propria involved inmaintaining mucosal homeostasis. Whether the observed effectsare due to one or a combination of microbial components is notknown. The analysis and characterization of the component(s)responsible for the observed effects are under scrutiny. This studysuggests that even noncommensal bacteria may have potent im-munomodulatory properties in mammals and that such bacteriamay represent a hitherto untapped and important source of mol-ecules relevant for studying mechanisms involved in homeostaticmicrobe-host interactions and in intestinal inflammation.

ACKNOWLEDGMENTS

Great thanks go to Rich Boden, University of Plymouth, for sharing hisknowledge about cultivation of Methylococcus capsulatus (Bath).

This work was supported by the Ostfold Hospital Trust.

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The Noncommensal Bacterium Methylococcus capsulatus (Bath) Ameliorates Dextran Sulfate (Sodium Salt)-Induced Ulcerative Colitis by Influencing Mechanisms Essential for Maintenance of the Colonic Barrier FunctionMATERIALS AND METHODSAnimals.Tissue collection.RNA extraction and cDNA synthesis.Real time-PCR.Histology.Cultivation of bacteria and preparation of M. capsulatus bacterial meal.Statistics.

RESULTSOral administration of bacterial meal to C57BL/6 mice reduced disease activity and histological inflammation in acute DSS-induced colitis.Inclusion of bacterial meal in the diet decreased intestinal inflammatory markers in mice with DSS-induced colitis.Neutrophil infiltration and activity were lower in mice fed a bacterial meal.In the colons of DSS-treated mice fed a bacterial meal, there was a lower induction of cytokine production.The bacterial meal-containing diet led to a slight increase in Muc2 mRNA expression.Mice fed the bacterial meal diet showed increased crypt depths and a higher number of proliferating epithelial cells.Oral administration of a bacterial meal of M. capsulatus (Bath) reduced disease activity and histological inflammation in acute colitis.

DISCUSSIONACKNOWLEDGMENTSREFERENCES

the noncommensal bacterium methylococcus capsulatus (bath ... · the noncommensal bacterium...

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